Discovery and in vitro evaluation of potent kinase inhibitors: Pyrido[1′,2′:1,5]pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2005.05.100

The discovery, synthesis, potential binding mode, and in vitro kinase profile of several pyrido[1′,2′:1,5]pyrazolo[3,4-d]pyrimidines as potent kinase inhibitors are discussed.

Full text of DOI:10.1016/j.bmcl.2005.05.100

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3778–3781
Discovery and in vitro evaluation of potent kinase inhibitors:
Pyrido[10,20:1,5]pyrazolo[3,4-d]pyrimidines
Michael J. Alberti, Elizabeth P. Auten, Karen E. Lackey, Octerloney B. McDonald,
Edgar R. Wood, Frank Preugschat, Geoffrey J. Cutler, Laurie Kane-Carson,
Wei Liu and David K. Jung*
GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA
Received 28 March 2005; revised 12 May 2005; accepted 16 May 2005
Available online 29 June 2005
Abstract—The discovery, synthesis, potential binding mode, and in vitro kinase profile of several pyrido[10,20:1,5]pyrazolo[3,4-d]-
pyrimidines as potent kinase inhibitors are discussed.
Ó 2005 Elsevier Ltd. All rights reserved.
Protein kinases catalyze the phosphorylation of tyrosine
and serine/threonine residues in various proteins in-
volved in the regulation of all functions.1 Protein kinases
can be broadly classified as receptor (e.g., EGFr,
c-erbB2, PDGFr, and VEGFR2) or non-receptor (e.g.,
c-src, b-raf, and ZAP70) kinases. Inappropriate or
uncontrolled activation of many of these kinases, by
over-expression, constitutive activation, or mutation,
has been shown to result in uncontrolled cell growth.2
Drug discovery efforts have targeted this aberrant kinase
activity in cancer, asthma, psoriasis, and inflammation,
to name a few.3
Recent advances in the identification of erbB family ki-
nase inhibitors have created hope for the modulation of
uncontrolled cell growth in cancer therapy for solid
tumors.4 For example, the compounds shown in
Figure 1, IressaTM and GW572016, continue to show
Figure 1. Successful examples of kinase inhibitors that have progressed
to clinical trials and patient care.
promising results in clinical trials on cancer patients.5
GleevecTMÕs activity in bcl-abl or c-kit-mediated malig-
Herein, we report the generation of a novel scaffold
nancies is well-documented and CEP1347 looks promis-
where the substitution pattern targets different regions
of the ATP-binding site of the protein kinase domain
ing for ParkinsonÕs disease.6,7 Despite these tremendous
results in the development of signaling inhibitors, there
to create differentially selective molecules. Based on lit-
remains a gap in the understanding of the selectivity
erature reports, linearly fused tricyclic core systems have
and required inhibition profile of kinase inhibitors to
been used as scaffolds for kinase inhibitors.9 Our goal
achieve efficacy without introducing toxicity.8
was to develop a novel tricyclic core ring system that
could be decorated with a variety of diverse substituents
to examine their structure–activity relationship (SAR)
against a panel of kinase inhibition assays. Herein, we
report the results of the utility of this approach to gen-
erate useful, selective tool compounds.
Keyword: Kinase inhibitor; VEGFR; GSK; Erb; EGFR.
*
Corresponding author. Tel.: +1 919 483 8391; fax: +1 919 483
6053; e-mail: david.k.jung@gsk.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.100

M. J. Alberti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3778–3781
3779
Scheme 3 depicts the sequence of reactions used to pre-
pare urea-substituted derivatives 6a–f. Subjecting the
aminonitrile intermediate 1 to microwave irradiation
in the presence of formamide results in the formation
of tricyclic amine 4. Stirring this product overnight with
an isocyanate in acetonitrile gave the desired N-phenyl-
N0-pyrido[10,20:1,5]-pyrazolo[3,4-d]pyrimidin-4-yl-urea
compounds. The synthetic sequences shown above are
amenable to the production of large number of com-
pounds by scaling up key intermediates 3 and 4, and
adding the final diversity in a parallel array format.10,11
Scheme 1. Synthesis of key intermediate 2-amino-3-cyano-pyrazol-
o[1,5-a]pyridine 1. Reagents: (a) malononitrile, ethanol, potassium
carbonate (25%).
Compounds 4, 5a–d, and 6a–f are representative exam-
ples of derivatives that can be synthesized by the meth-
ods described above. The compounds were evaluated in
a panel of kinase enzyme assays and the data for erbB2,
EGFR, GSK3, and VEGFR2 are summarized in Tables
1 and 2. The free amino-substituted derivative 4 showed
no activity against EGFR, erbB2, GSK3, or VEGFR.
However, replacement of the amino substituent with
aniline derivatives resulted in potent inhibitors of erbB2
and/or EGFR, with selectivity over GSK3 and VEGFR.
The SAR that confers potency and selectivity to the
quinazoline series for the erbB family. TK inhibition
was evaluated in N-phenylpyrido[10,20:1,5]pyrazolo[3,4-
d]pyrimidin-4-amines.12 For example, 5a has a ÔsmallÕ
anilino group, and like IressaTM, is selective for EGFR
Scheme 2. Synthesis of N-phenylpyrido[10,20:1,5]pyrazolo[3,4-d]pyrim-
idin-4-amine derivatives 5a–d. Reagents and conditions: (a) formic
acid, cat. sulfuric acid, 90 °C; (b) POCl3 (61%, two steps); (c) anilines,
i-PrOH, reflux.
Table 1. N-Phenylpyrido[10,20:1,5]pyrazolo[3,4-d]pyrimidin-4-amines
Compound
Structure
ErbB214
EGFR14
Scheme 3. Synthesis of N-phenyl-N0-pyrido[10,20:1:5]pyrazolo[3,4-
d]pyrimidin-4-yl-urea derivatives 6a–f. Reagents and conditions: (a)
formamide, MW 240 °C (79%); (b) isocyanates, MeCN, 25 °C, 16 h.
4
>10
>10
The syntheses of two novel classes of kinase inhibitors, N-
phenyl-pyrido[10,20:1,5]pyrazolo[3,4-d]-pyrimidin-amine
and N-phenyl-N0-pyrido[10,20:1,5]pyrazolo [3,4-d]-pyrim-
idin-4-yl-urea derivatives, are shown in Schemes 1–3.
5a
5b
>10
0.200
0.032
The key aminonitrile intermediate 1 was synthesized by
combining the commercially available N-aminopyridini-
um iodide and malononitrile in ethanol. Heating this
mixture with microwave irradiation in the presence of
2 equiv of potassium carbonate generates the desired
product. This intermediate has been used to prepare
two classes of kinase inhibitors. Preparation of those
derivatives with aniline substituents at the 4-position is
shown in Scheme 2, and a synthetic approach used to
prepare inhibitors with urea substitution at the 4-posi-
tion is shown in Scheme 3.
0.063
5c
5d
0.200
0.050
The synthesis of anilino-substituted analogs was started
by heating the aminonitrile 1 with formic acid in the
presence of catalytic sulfuric acid (Scheme 2). The result-
ing pyrimidinone derivative 2 was converted to the corre-
sponding chloroimidate 3 with phosphorous oxychloride.
The final displacement of the chloride with an appropriate
aniline was accomplished by heating in isopropyl
alcohol to give the desired N-phenylpyrido[10,20:1,5]pyr-
azolo[3,4-d]pyrimidin-4-amine compounds (5a–d).
1.58
5.01
Kinase enzyme inhibition expressed as IC50 values in micromolar.14
The IC50 values are >10 lM for GSK3 and VEGFR2 for compounds 4
and 5a–d.

3780
M. J. Alberti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3778–3781
Table 2. N-Pyrido[10,20:1,5]pyrazolo[3,4-d]pyrimidin-4-yl-ureas
Compound
Structure
GSK315
0.126
VEGFR16
6a
0.200
6b
6c
6d
6e
6f
0.158
0.316
1.00
0.251
>10
Figure 2. Docking model of 5b (green), compared with a cocrystal of
GW572016 (purple) in EGFR.8 Hydrogen bonds are indicated by
dashed lines.
3.98
1.58
3.98
>10
0.631
Kinase enzyme inhibition expressed as IC50 values in micromolar.15,16
The IC50 values are >10 lM for ErbB2 and EGFR for compounds
6a–f.
Figure 3. Docking model of 6a (yellow), compared with a cocrystal of
GW572016 (purple) in EGFR.8 Hydrogen bonds are indicated by
dashed lines.
over erbB2. While a larger aniline substituent in 5b, like
GW572016, is a potent dual EGFR/erbB kinase inhibi-
tor (Fig. 2). The potency of this dual inhibition is depen-
dent on substitution on the aniline moiety, and a drop in
potency was observed with 5c and d.
there is space for 6- and 7-position substitutions for in-
creased interactions.
When the aniline substituent of a pyrido[10,20:1,5]pyraz-
olo[3,4-d]-pyrimidine scaffold is changed to an aryl urea,
no activity against erbB2 or EGFR is observed. This
lack of erbB2/EGFR inhibition from the urea deriva-
tives may be attributed to the fact that a threonine res-
idue exists in the ATP-binding pocket of EGFR and
erbB2. This threonine (Thr830) forms a water-mediated
hydrogen bond from the quinazoline N-3 nitrogen of
GW572016 to the threonine hydroxyl (see Fig. 2). This
same interaction could exist with the aniline-substituted
pyrido[10,20:1,5]pyrazolo[3,4-d]pyrimidines.
To confirm our belief that the novel tricyclic ring system
was able to bind in the ATP-binding site of EGFR so
that it possessed similar SAR to the quinazolines, we
modeled several viable binding modes. The best model
is shown in Figure 2 where 5b is compared with a co-
crystal structure of GW5720168. The aniline portion of
the compounds is overlaid in an identical fashion in
the back-pocket region, allowing the N-1 to interact
with the hinge region. The tricyclic core then extends
down the center of the binding site, which suggests that

M. J. Alberti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3778–3781
3781
13. Cherry, M.; Williams, D. H. Curr. Med. Chem. 2004, 11,
663.
However, interaction with the urea-substituted deriva-
tives is disrupted by the existence of an intramolecular
hydrogen bond between the urea nitrogen and the corre-
sponding nitrogen of the tricyclic core (see Fig. 3).13
This intramolecular hydrogen bond not only disrupts
the interactions with the threonine of EGFR/erbB2,
but also slightly changes the relative orientation of the
aryl substituents of the aniline and urea derivatives
when superimposed on each other.
14. Compounds were tested for EGFR or ErbB-2 protein
tyrosine kinase inhibitory activity in substrate phosphor-
ylation assays using enzymes purified from a baculovirus
expression system. Reagent production and assay meth-
odology were conducted essentially as described (Brignola,
P. S. et al. J. Biol. Chem. 2002, 277, 1576). The method
measures the ability of the isolated enzyme to catalyze the
transfer of the c-phosphate from ATP onto tyrosine
residues in a biotinylated synthetic peptide (biotin-Ahx-
RAHEEIYHFFFAKKK-amide). Reactions were per-
formed in 96- or 384-well polystyrene plates in a final
volume of 20 or 45 ll. Reaction mixtures contained
50 mM MOPS (pH 7.5), 2 mM MnCl2, 10 lM ATP,
0.125 lCi [c-33P]ATP per reaction, 2 lM peptide sub-
strate, and 1 mM dithiothreitol. Reactions were initiated
by adding 1 pmol (20 nM) per reaction of the indicated
enzyme. The reaction was allowed to proceed for 15 min,
terminated, and quantified using a scintillation proximity
assay procedure as described (McDonald, O. B., Antons-
son, B., Arkinstal, S., Marshall, C. J., and Wood, E. R.
Anal. Biochem. 1999, 268, 318).
15. The catalytic domain of vascular endothelial growth
factor receptor 2 (VEGFR2) was expressed and purified
using methods similar to those described for ErbB-2 and
EGFR. Kinase assays were performed as described above
with the following modifications: VEGFR2 assays con-
tained 10 nM enzyme, 100 mM HEPES, pH 7.5, 0.1 mg/ml
bovine serum albumin, 0.1 mM dithiothreitol, 360 nM
peptide A, 75 lM ATP, and 5 mM MgCl2. The reaction
was allowed to proceed for 40 min. Product was detected
using a homogeneous time-resolved fluorescence proce-
dure (Park, Y.-W., Cummings, R. T., Wu, L., Zheng, S.,
Cameron, P. M., Woods, A., Zaller, D. M., Marcy, A. I.,
and Hermes, J. D. Anal. Biochem. 1999, 269, 94). Briefly,
the reactions were quenched by adding 100 ll of 100 mM
HEPES, pH 7.5, 100 mM EDTA, 45 nM streptavidin-
linked allophycocyanin (Molecular Probes, Eugene, OR),
and 3 nM europium-conjugated anti-phosphotyrosine
antibody (Wallac, Turku, Finland). The product was
detected using a Victor plate reader (Wallac, Turku,
Finland) with a time delay at 665 nm.
Although the urea-substituted pyrido[10,20:1,5]pyrazol-
o[3,4-d]pyrimidines do not show inhibition of EGFR/
erbB2, they do begin to show moderate inhibition
against GSK3 and/or VEGFR2 (see Table 2). The range
of potencies within the urea-substituted series provides
SAR which suggests that this template could be used
to develop GSK3 selective compounds (e.g., 6c) or
VEGFR2 selective compounds (e.g., 6f).
The facile synthesis and kinase inhibition data for the
pyrido[10,20:1,5]pyrazolo[3,4-d]-pyrimidine derivatives
demonstrate the potential of this scaffold to generate di-
verse kinase inhibition profiles. The most obvious trend
observed is that the anilino-substituted derivatives dem-
onstrated inhibition of the erbB family, but not GSK3
or VEGFR2, while the reverse trend is observed for
the urea-substituted derivatives.
References and notes
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S(PO3)YR-amide, was detected using
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Biotin-Ahx-AAAKRREILSRRP-
scintillation
a
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proximity assay (SPA) method as described above. Assay
conditions were as follows: 100 mM HEPES, pH 7.2,
10 mM MgCl2, 0.3 mg/mL heparin sulfate, 0.1 mg/mL
BSA , 1 mM DTT , 2.5 lM ATP, 0.6 uCi/rxn 33P labeled
ATP, and 1.2 lg/mL GSK-3b protein. The plates are
incubated at room temperature for 19 min prior to the
addition of SPA stop solution.